OBJECTIVETo establish a new method for screening active ingredients of Chinese herbs by determining different bio-thermodynamic effects of 3 genosides on splenic lymphocyte of mice.

METHODSUsing a thermal bioactivity monitoring system, the maximum heat output (mHO), average metabolic heat (MH) and constant of decrease rate (DR) of lymphocyte were determined based on the growth metabolic power-time curve, and the outcomes were verified by MIT.

RESULTSThe mHO and MH increased and the DR decreased after lymphocytes being exposed to the 3 genosides in different concentrations, arranged upon their potency as genoside Rg3 > genoside Rg2 > genoside Rg1 (merely insignificant effect). MTT showed the same results.

CONCLUSIONHeat activity monitoring system could precisely display the different bio-thermal dynamic effects of 3 genosides on splenic lymphocyte.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Energy Metabolism ; drug effects ; Ginsenosides ; pharmacology ; Lymphocytes ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; Thermodynamics

AIMTo investigate effects of hsBAFF synthesized in Escherichia coli on spleen B lymphocyte immune response and its intracellular free Ca2+ ([Ca2]i]) signaling in mice.

METHODSTwenty ICR mice, half males-half females, were chosen and randomly divided into a normal control group (n=10) and a hsBAFF treatment group (n-10). The mice in hsBAFF treatment group were given abdominal cavity injection of hsBAFF solution which was diluted with phosphate buffered saline (PBS) at dosage of 0.1 mg/kg body weight once each day for over eight days. The mice in control group were received abdominal injection of PBS at the same dose and frequency. Spleen B lymphocyte proliferation and its immune response to LPS stimulation in mice were evaluated using an MTT assay, and change of spleen B lymphocyte [Ca2+]i was assayed under a laser scanning confocal microscope.

RESULTSB lymphocyte proliferation and its immune response to LPS stimulation were significantly higher in hsBAFF-treated mice than in control mice (P < 0.05). The B lymphocyte [Ca2+]i fluorescence intensity in hsBAFF-treated mice maintained at a relatively high level fluctuation, and its average intensity was significantly higher to that of control mice (P < 0.01), but change rate of the intensity was lower compared to that of control group.

CONCLUSIONhsBAFF synthesized in Escherichia coli can enhance immune function in the body by increasing B lymphocyte proliferation and its immune response. hsBAFF-activated B lymphocyte function may be associated with increasing B lymphocytes [Ca2+]i.

Animals ; B-Cell Activating Factor ; immunology ; pharmacology ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Proliferation ; drug effects ; Female ; Male ; Mice ; Mice, Inbred ICR ; Spleen ; cytology ; drug effects ; immunology

OBJECTIVETo investigate the protection of Hsian-tsao(Mesona procumbens Hemsl)on H(2)O(2)-induced DNA damage of primarily cultured spleen lymphocytes.

METHODSSpleen lymphocytes were primarily cultured for 24 h and then divided into 6 groups randomly: 4 groups were treated with different concentrations of Hsian-tsao extracts for 60 min, then with 50 micromol/L H(2)O(2); 1 group was treated with 50 micromol/L H(2)O(2) only and the last group with PBS. Twenty min after H(2)O(2)or PBS treatment SCGE and laser confocal microscopy were performed, then the comet length and percentage of cells with migrated DNA were measured.

RESULTSH(2)O(2) caused severe DNA damage in primarily cultured spleen lymphocytes and Hsian-tsao extracts reduced the DNA damage at a concentration of 10, 50 and 100 microg/ml. The quantity of comet cells was reduced from 100% to 88%, 60%, 36% (P<0.05, 0.01, 0.01) and the comet length was reduced from (49.56+/-6.94) microm to (41.14+/-5.64) microm, (38.89+/-9.81) microm, (28.62+/-4.66) microm (P<0.05, 0.01, 0.01), respectively.

CONCLUSIONHsian-tsao has dramatic ability of antioxidant. It can protect cells from the damage of ROS-induced DNA injury in the certain range.

Animals ; DNA Damage ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hydrogen Peroxide ; Lamiaceae ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Protective Agents ; pharmacology ; Spleen ; cytology

OBJECTIVETo discuss the effect of Yisui Shengxue granules on expression of alpha-hemoglobin stabilizing protein (AHSP) mRNA in different developmental stages mice.

METHODThe total RNAs were extracted from the bone marrow karyocyte of normal adult mice and the karyocyte of fetus liver and fetus spleen in pregnanted mice (pregnanted 21 days) and fetal mice (pregnanted 14 days). The expression level of AHSP mRNA in different developmental stages mice interfered with Yisui Shengxue granules was measured by real-time PCR.

RESULTThe intervention of Yisui Shengxue granules could significantly up-regulated the expression levels of AHSP mRNA in normal adult mice.

CONCLUSIONThe result revealed that one of possible molecular mechanism of the effects caused by Yisui Shengxue granules is that it can promote the AHSP gene expression, reduce the free a-globin deposit, then prevent the poison to erythrocyte and decrease the haemolysis.

Animals ; Blood Proteins ; genetics ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Erythrocytes ; cytology ; drug effects ; metabolism ; Female ; Gene Expression Regulation, Developmental ; drug effects ; Liver ; cytology ; embryology ; metabolism ; Male ; Mice ; Molecular Chaperones ; genetics ; Plants, Medicinal ; chemistry ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Spleen ; cytology ; embryology ; metabolism ; Up-Regulation ; drug effects ; genetics

OBJECTIVETo study the effect of Chinese drugs for invigorating qi and tonifying Shen (IQTS) on expression of CD4+CD25+ regulatory T cells in spleen and maternal-fetal interface of abortion-prone mice during pregnancy.

METHODSCBA female mice were mated with DBA/2 male mice to establish abortion-prone models, which were randomly divided into 4 groups, the negative control group (fed with normal saline), the positive control group (treated with CsA), the Chinese medicine group (treated with IQTS), and the Chinese and Western medicine group (treated with IQTS+CsA). Mice were sacrificed in batches on the 9th and the 14th day of gestation, their splenic and decidual tissues were taken out to analyse CD4+CD25+ regulatory T-cell expression by flow cytometry.

RESULTSCompared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 9th day of gestation (P < 0.01 or P < 0.05). There was no statistical difference in intergroup comparison of the three treatment groups (P > 0.05). Compared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 14th day of gestation (P < 0.01 or P < 0.05). Of them, its expression was the highest in the Chinese and Western medicine group, showing significant difference from that in the Chinese medicine group and the positive group (P < 0.01). The difference between the Chinese medicine group and the positive group was insignificant (P > 0.05). On day 9 of gestation, compared with the negative control group, the expressions of CD4+CD25+ regulatory T in maternal-fetal interface increased in the three treated groups, showing no statistical significance (P > 0.05). Its expression was ordered from high to low in sequence as the Chinese and Western medicine group, the positive control group, the Chinese medicine group, and the negative control group. On day 14 its expression was obviously enhanced in the Chinese and Western medicine group, showing statistical difference from that in the negative control group (P < 0.05). But its expression was obviously enhanced in the Chinese medicine group and the positive group, showing insignificant difference from that in the negative group. The same sequence was found in the percentage of CD4+CD25+ T cells in CD4+ T cells.

CONCLUSIONSChinese drugs for IQTS could up-regulate the expression of CD4+CD25+ regulatory T in spleen of abortion-prone mice in the early and late pregnancy stages. When combined with CsA, it also could up-regulate its expression in maternal-fetal interface in the mid and late pregnancy stages, suggesting that Chinese drugs for IQTS are facilitate to maintain the immune tolerance state in mice during pregnancy.

Abortion, Spontaneous ; drug therapy ; immunology ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Male ; Mice ; Mice, Inbred CBA ; Phytotherapy ; Pregnancy ; Spleen ; cytology ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.
Animals ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cordyceps ; Immune Tolerance ; Lymphocyte Activation ; drug effects ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Polysaccharides ; pharmacology ; Spleen ; cytology ; Weightlessness Simulation

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 μg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 μg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.
Animals ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Phenanthrenes ; pharmacology ; Signal Transduction ; Spleen ; cytology ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

OBJECTIVETo evaluate the effect of Herba Epimedii Brevicornus (HEB) and prepared Radix Rehmannia (RR), the representative Chinese herbs for warming yang and nourishing yin on glucocorticoid receptor (GR) in GR down-regulated rats.

METHODSThe GR down-regulated model was established by subcutaneous injection of hydrocortisone. Seventy-two SD male rats were randomly divided into four groups: the normal group; the model group (MG) ; the HEB group, and the RR group. And every group was subdivided to 3 batches depending on the time points (the 3rd, 7th and 14th day after modeling) of observation, with 6 rats in each batch. Changes in blood level of corticosterone (GS), as well as protein expression and binding power of GR in splenic lymphocytes at corresponding time points of the batches were determined by RIA and flow cytometry.

RESULTSNo significantly difference was found in blood GS levels of the model group at all the time points, as compared with that of the normal group (P > 0.05), while that in the RR group was significantly lowered at the 14th day, and showed significant difference from that in the model group (P < 0.05). The protein expression and binding power in the model rats were lower than those in normal ones (all P < 0.01), but they were significantly higher on the 7th and 14th day in the RR group (P < 0.01), and on the 14th day in the HEB group higher than those in the model group, respectively (all P < 0.01).

CONCLUSIONBoth HEB and RR, in dosage-form of water decoction, could up-regulate the protein expression and binding power of GR in GR down-regulated model rats, but different in acting time.

Animals ; Corticosterone ; blood ; Disease Models, Animal ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Flow Cytometry ; Hydrocortisone ; pharmacology ; Lymphocytes ; cytology ; drug effects ; metabolism ; Male ; Radioimmunoassay ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; metabolism ; Rehmannia ; chemistry ; Spleen ; cytology

OBJECTIVETo investigate the effect of Guilu Erxianjiao (GLEXJ) in suppressing chemotherapy induced cell apoptosis in mice and to discuss its possible acting mechanism.

METHODSSeventy-two tumor-bearing Kunming mice were randomly divided into 6 groups, Group A, treated with normal saline; Group B treated with cyclophosphamide (CTX), Group C treated with Chinese herbal medicine, and Group D-F, the three combined treated group received CTX plus high, moderate and low dose GLEXJ. All mice were sacrificed after 9-day medication, their splenic tissues were taken for determining T-lymphocyte apoptosis with flow cytometer, the expression of bcl-2 mRNA and Caspase-3mRNA detected by RT-PCR.

RESULTSAs compared with Group A, FITC+/PI- cell proportion of spleen T-lymphocyte cells and Caspase-3mRNA expression were higher, while bcl-2 mRNA expression was lower in Group B; as compared with Group B, the former two indexes were lower and the latter was higher in the three combined treated groups (all P <0.05).

CONCLUSIONGLEXJ could efficiently suppress the splenic T-lymphocyte apoptosis in tumor bearing mice undergoing chemotherapy, one of its mechanisms may be through up-regulating of bcl-2 mRNA expression and down-regulating of Caspase-3mRNA expression.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Mice ; Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Random Allocation ; Spleen ; cytology ; drug effects ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the influence of high mobility group box-1 protein (HMGB1) on the immunosuppression function of splenic regulatory T cells (Tregs) and its potential regulatory mechanism underlying the effect on CD4+ CD25- T cells in mice.

METHODSCD4+ CD25+ Tregs isolated from the spleens of male BALB/c mice by magnetic beads were seeded on 96-well (1 x 10(5) cells/well) cell culture plates coated with 1 microg/ml anti-CD3 and soluble CD28. After being stimulated with HMGB1 for different time and concentrations, the secretions of IL-2 and IL-10 were analyzed by ELISA. Tregs stimulated for 72 hours were cultured with CD4+ CD25- T cells together. The suppressive activity of CD4+ CD25+ Treg to CD4+ CD25- T cells was analyzed by MTT test. IL-2, IL-10, IL-4, and interferon (IFN)-gamma in the cell suspensions were determined by ELISA.

RESULTSAfter stimulation with HMGB1, the suppressive activity of splenic Tregs in mice were significantly down-regulated at 72 hours, when the proportion of Tregs to CD4+ CD25- T cells was 1 : 1. The secretion of IL-2 of Tregs stimulated by HMGB1 was not markedly changed (P > 0.05), while a dose-dependent decrease between IL-10 induction and HMGB1 concentration was obviously (P < 0.05). When CD4+ CD25- T cells were cultured with stimulated Tregs, comparing with unstimulated-Treg group, levels of IL-2 and IFN-gamma were elevated following the increased concentration of HMGB1 (P < 0.05 or P < 0.01). Meanwhile the secretion of IL-4 and IL-10 significantly decreased when cultured with stimulated Tregs (P < 0.05).

CONCLUSIONSThese data suggested that HMGB1 stimulation can result in significant down-regulation of immunosuppression of splenic Tregs in mice. HMGB1 might be a potential immunoregulatory signal that influences the proliferation of effector T cells, secretion of IL-2 and cells-polarization by inhibiting CD4+ CD25+ Tregs activity.

Animals ; Cell Communication ; drug effects ; Cells, Cultured ; HMGB1 Protein ; pharmacology ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Receptors, Interleukin-2 ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism